Minocycline effects on IL-6 concentration in macrophage and microglial cells in a rat model of neuropathic pain

Background: Evidence indicates that neuropathic pain pathogenesis is not confined to changes in the activity of neuronal systems but involves interactions between neurons, inflammatory immune and immune-like glial cells. Substances released from immune cells during inflammation play an important role in development and maintenance of neuropathic pain. It has been found that minocycline suppresses the development of neuropathic pain. Here, we evaluated the analgesic effect of minocycline in a chronic constriction injury [CCI] model of neuropathic pain in rat and assessed IL-6 concentration from cultured macrophage and microglia cells

Methods: Male Wistar rat [n=6, 150-200 g] were divided into three different groups: 1] CCI+vehicle, 2] sham+vehicle, and 3] CCI+drug. Minocycline [10, 20, and 40 mg/kg] was injected one hour before surgery and continued daily to day 14 post ligation. Von Frey filaments and acetone, as pain behavioral tests, were used for mechanical allodynia and cold allodynia, respectively. Experiments were performed on day 0 [before surgery] and days 1, 3, 5, 7, 10, and 14 post -injury. At day 14, rats were killed and monocyte-derived macrophage from right ventricle and microglia from lumbar part of the spinal cord were isolated and cultured in RPMI and Leibovitz's media, respectively. IL-6 concentration was evaluated in cell culture supernatant after 24 h

Results: Minocycline [10, 20, and 40 mg/kg] attenuated pain behavior, and a decrease in IL-6 concentration was observed in immune cells compared to CCI vehicle-treated animals

Conclusion: Minocycline reduced pain behavior and decreased IL-6 concentration in macrophage and microglial cells

Formulation development and toxicity assessment of triacetin mediated nanoemulsions as novel delivery systems for rapamycin

The aim of this investigation was to design and develop nanoemulsions [NEs] as novel delivery systems for rapamycin. Phase behavior of quaternary systems composed of Traicetin [as oil], various surfactants and co-surfactants and water at different surfactant/co-surfactant weight ratios was investigated by the construction of phase diagrams. Formulations were taken from the o/w NE region of the phase diagrams, depending upon the extent of NE domain. The spontaneous emulsification method was used to prepare various formulations containing 1 mg/mL of the drug. The NEs were characterized and subjected to stability tests at various temperatures over 9-12 months. Cumulative drug release from the selected formulations was determined for a period of 48 h using a dialysis sac. The assay of rapamycin was carried out using an HPLC technique. The effect of NEs on the viability of SKBR-3 cells was evaluated by MTT assay. The integrity of Caco-2 cell monolayers was measured by Transepithelial Electrical Resistance [TEER] and the transport of rapamycin-loaded NEs across Caco-2 cell monolayers was then assessed. The uptake of NEs by SKBR-3 cells was also investigated using florescence microscopy. Maximum drug release was observed in case of 4 formulations prepared with Tween 80 and Tween 20. MTT test results revealed different toxicity of NEs for SKBR-3 cell line and TEER demonstrated that formulations containing Tween 20 caused a more considerable decrease in cell integrity in comparison with those prepared with Tween 80. The results obtained from cellular uptake experiments were in consistent with those obtained from TEER and cytotoxicity experiments

Investigating the effect of Phlomis lanceolata boiss and hohen on cancer cell lines

Phlomis lanceolata is a medicinal plant that has long been used to treat various conditions such as diabetes, gastric ulcer, hemorrhoids, inflammation and wounds. As most of Phlomis species have shown cytotoxic activity against proliferation of different cell lines, a biological investigation of P. lanceolata was carried out in this study. The aim of this study was to find out the in vitro cytotoxic activity of total extract and different fractions of Phlomis lanceolata on four cell lines. Cytotoxic activity of the metanolic total extract and partition fractions of chloroform, ethyl acetate and petroleum ether of flowering aerial parts of Phlomis lanceolata on the HT29, Caco2, T47D and NIH3T3 cell lines is examined by MTT. Petroleum ether fraction showed high cytotoxic activity against proliferation of all four cell lines. Presence of heavy triterpens and lipophil compounds recognized by TLC test in Petroleum ether fraction is responsible for high cytotoxic activity. The results emphasize the importance of phytochemical studies which could lead to the discovery of new active compounds

Long acting propranolol and HSP-70 rich tumor lysate reduce tumor growth and enhance immune response against fibrosarcoma in Balb/c mice

Noradrenaline [NA], the principal neurotransmitter released from sympathetic nerve terminals, influences T-cell maturation, not only directly in developing T cells, but also indirectly, by acting on the thymic nonlymphoid cells. In vitro and in vivo studies have demonstrated the anti-proliferative, anti-migratory, antiangiogenic and cytotoxic properties of propranolol, beta-AR blocker, against various cancers. To evaluate the effect of propranolol on efficacy of HSP-70 rich lysate vaccine in immunotherapy of fibrosarcoma. Mouse fibrosarcoma WEHI-164 cells were used to immunize tumor-bearing mice with or without propranolol and HSP-70. Splenocytes proliferation, cytotoxic activity of the splenocytes, naturally occurring CD4+ CD25[high] T-reg cells and IFN-gamma and IL-4 secretion as well as tumor size, were assessed to describe the anti-tumor immune response. A significant increase in the level of IFN- gamma in the mice vaccinated with WEHI-164 cells enriched with HSP-70 and co-treated with propranolol was observed compared to controls. However, HSP enrichment or propranolol treatment alone did not enhance the immune response as measured by the level of IFN-gamma. Likewise, a decrease in tumor growth in the test group [p<0.01] and a significant increase in CTL activity [p<0.05] was observed. HSP enriched vaccine shows anti-tumor activity, probably due to the modulation of immune responses

Neuroprotective effect of total and sequential extract of Scrophularia striata boiss. in rat cerebellar granule neurons following glutamate-induced neurotoxicity: an in-vitro study

Neuroprotective effect of the extract from aerial parts of Scrophularia striata Boiss [Scrophulariaceae] was investigated against glutamate-induced neurotoxicity on cultured rat pups Cerebellar Granule Neurons [CGNs]. CGNs from 8 days old Sprague-Dawley rat were prepared and cultured. The experiments were performed after 8 days in culture. The plant was collected from the northeastern part [Ruin region] of Iran and air-dried at room temperature. The total extract was prepared with maceration of prepared powder in ethanol 80% for three times. Sequential extracts were obtained using dried and powdered aerial parts with increasingly polar solvents: petroleum ether, chloroform, ethyl acetate and methanol 80% solution. Cultured cells were exposed to 125 micro M of glutamate for 12 h following a 24 h of incubation with test fractions at concentration of 10 mcg/mL. Morphological assay was performed using invert light microscope after fixation and staining with haematoxylin. Neuronal viability was measured using MTT assay. Statistical analysis was done using SPSS software. One way analysis of variance [ANOVA] was performed by Tukey post-hoc test. Values were considered statistically significant when p-value < = 0.05. Results of this study showed a significant neuroprotective activity of high polarity methanolic fraction of aerial parts of Scrophularia striata against glutamate-induced neurotoxicity in a dosedependent manner. Treatment with 10 mcg/mL of the fractions showed the best result

P53 but not cyclin E acts in a negative regulatory loop to control HER-2 expression in MCF-7 breast carcinoma cell line

Cyclin E, HER-2 and p53, are considered as major prognostic markers in breast cancer. As they are related in patho-clinical level, we aimed to check if they have any direct interaction on expression of each other. To study the effect of cyclin E on HER-2 expression, cell lines stably overexpressing cyclin E or its low molecular weight [LMW] isoforms were generated. To understand the results of p53 silencing either alone or in combination with cyclin E overexpression, we created three different p53 stably knocked down cell lines. Protein expression was analyzed by western blot, HER-2 expression in the established cell lines were determined using SYBR green real time PCR and data analyzed by REST software. Results indicate that HER-2 expression is only downregulated following p53 silencing and none of cyclin E isoforms can alter its expression. The presence of cyclin E isoforms in p53 silenced clones also does not altered HER-2 expression. Given the fact that p53 degradation is increased by HER-2 overexpression, these data can draw a regulatory loop in which a non-mutated functional p53 and HER-2 can bidirectionally regulate the expression of these two genes. This study improves our understandings of these pathways and these proteins can be introduced either as a marker or as a target in cancer treatment.

time course of JNK and P38 activation in cerebellar granule neurons following glucose deprivation and BDNF treatment

Low glucose condition induces neuronal cell-death via intracellular mechanisms including mitogen-activated protein kinases [MAPK] signaling pathways. It has been shown that low glucose medium decreases neuronal survival in cerebellar granule neurons [CGNs]. In this study, we have examined the activation of JNK, p38kinase and ERK1/2 pathways in low glucose medium in CGNs. The CGNs were prepared from new-born [P-2 and P-5] rats and cultured in Dulbecco's Modified Eagle's Medium high [DMEM-HIGH] glucose supplemented with Fetal Bovine Serum [FBS] 10% for 7 days. The glucose deprivation was induced through replacing the culture medium with the low glucose [5 mM] medium. The MAPK pathways activation was evaluated through phospho specific antibodies using western blot. The viability of cells was measuring using MTT assay. The results indicated that low glucose reduces the cell survival and brain-derived neurotrophic factor [BDNF] elevates the cell viability in CGNs. The basal c-Jun N-terminal kinase [JNK] activity was high in CGNs and glucose deprivation for 24 h had increased phospho-JNK level to 2-fold compared to basal. BDNF treatment reduced the basal JNK activity within 30 min but had no effect in longer incubations. BDNF also blocked the low glucose-induced JNK activation. In addition, CGNs exhibited high p38 phosphorylation in low glucose medium in 48 h. These results demonstrated that in sustained low glucose conditions, CGNs had high activity of stress-activated MAPK which could induce cellular damage. Moreover, BDNF can prevent JNK and p38 activation in stress conditions and increase cell viability. Our results suggest that in sustained stress conditions, inhibition of JNK and/or p38 pathways might protect neurons from damage in low glucose conditions

comprehensive review of punica granatum [pomegranate] properties intoxicological, pharmacological, cellular and molecular biology researches

Punica granatum [Pg], commonly known as pomegranate [Pg], is a member of the monogeneric family, Punicaceae, and is mainly found in Iran which is considered to be its primary centre of origin. Pg and its chemical components possess various pharmacological and toxicological properties including antioxidant, anti-inflammatory [by inhibiting pro-inflammatory cytokines], anti-cancer and anti-angiogenesis activities. They also show inhibitory effects on invasion/motility, cell cycle, apoptosis, and vital enzymes such as cyclooxygenase [COX], lipooxygenase [LOX], cytochrome P450 [CYP450], phospholipase A2 [PLA2], ornithine decarboxylase [ODC], carbonic anhydrase [CA], 17beta-hydroxysteroid dehydrogenase [17beta-HSDs] and serine protease [SP]. Furthermore, they can stimulate cell differentiation and possess anti-mutagenic effects. Pg can also interfere with several signaling pathways including PI3K/AKT, mTOR, PI3K, Bcl-X, Bax, Bad, MAPK, ERK1/2, P38, JNK, and caspase. However, the exact mechanisms for its pharmacological and toxicological properties remain to be unclear and need further evaluation. These properties strongly suggest a wide range use of Pg for clinical applications. This review will discuss the areas for which Pg has shown therapeutic properties in different mechanisms

Effects of continuous and interrupted forces on gene transcription in periodontal ligament cells in vitro

The biological mechanisms of tooth movement are based on the response of periodontal tissues to mechanical forces. The final result of these responses is remodeling of the extracellular matrix. Tissue reactions may vary depending upon the type, magnitude and duration of the applied forces. The purpose of the present study was to analyze the effects of centrifugal force on the transcription of collagen type-I [Col-I], matrix metalloproteinase-1 [MMP-1], and tissue inhibitor of metalloproteinase-1 [TIMP-1] genes in human periodontal ligament [PDL] fibroblasts. Human fibroblasts obtained from the PDL were cultured and subjected to centrifugal forces [36.3 g/cm2] for 30, 60 and 90 min continuously. This was also carried out interruptedly, three times for 30 min and six times for 15 min. The mRNAs encoding for Col-I, MMP-1, and TIMP-1 were quantified using RT-PCR. The mRNA levels of Col-I and MMP-1 were increased when continuous force was applied for 30 min and 60 min respectively. The interrupted force had almost no effect on Col-I, MMP-1 and TIMP-1 genes. These results indicate that continuous forces may have a greater effect in inducing gene expression during the remodeling process of PDL compared to interrupted forces with short rest periods

Lead exposure impairs NMDA agonist-induced no production in pyramidal hippocampal cells

Chronic exposure to Lead [Pb] affects neural functions in central nervous system [CNS] particularly the learning and memory. On the other hand, alteration of calcium level in the CNS results in activation of NOS where it is expected to increase nitric oxide level in hippocampus. In this study the role of Lead exposure in NMDA induced NO production in pyramidal hippocampal cells [CA1HP] was investigated. The NO level was determined by measurement of concentration of nitrite and nitrate as NO products using the metHb production at 401 nm. The ACBD [NMDA agonist]-induced NO level was almost reduced to the control level [2.5 nM] in the presence of 10 and 100 nM of Lead acetate. Lead acetate at concentrations which normally results in chronic toxicity did not increase the nitric oxide [NO] production by CA1HP. One reason for this finding could be the interaction of Lead with NMDA receptors due to similarity of Pb[2+] to Zn[2+] ion. Another reason may be related to direct interaction of Lead with NMDA receptors that inhibit the stimulated NO production

Evaluation of potency of measles vaccine used in Iran: comparison of WHO and NIBSC method in cell culture

Measles has been a major cause of illness and death in children and vaccination against the disease is part of the WHO global immunization program. A suitable vaccine should create maximum immune response against the pathogen and must be safe for the user. Thus, after production, vaccines must be analyzed and controlled by the producer and confirm by relevant governmental organizations. The Food and Drug Control Lab [FDCL], Ministry of Health, is the secondary control center on potency of vaccines in Iran. In this study, we have set up the WHO and NIBSC methods in FDCL and compare these methods on determining the potency of measles vaccine. Measles vaccines were obtained from Razi Institute Iran. Nine dilutions of vaccine [10[-1] to 10[-5]] in 0.5 log interval were mixed with Vero cell suspension and seeded. In WHO method, the cells were incubated at 36°C for 10 days, during which the cells were checked for cytopatic changes everyday. To set up the assay, we tested the vaccine dilution with four different cell suspensions [2x10[5]-5x10[4]/well] and four different concentration of serum [2.5-10%]. Based on our results, in the assays, 5% serum and 1x10[5] cells were used. The potency assay was performed with six different vaccines produced in one batch and the mean potency for Measles was 10[4.32 +/- 0.24] CCID[50]/vial for a ten-dose vial. In NIBSC method following seeding of Vero cells, the medium was removed after 3 hours and overlay was added. Then the plates were incubated at 35°C for 10 days. After incubation period, the overlay was removed, the plaques were stained with methyl violet and counted. This assay was repeated three times and the mean of the results was 5.83 +/- 0.03 log[10] PFU/dose. In this study, we have set up the WHO and NIBSC methods and results indicated that the potency of the vaccine is in acceptable range in either method. Furthermore, the WHO method is simple and less time consuming compared to NIBSC which is complicated and requires more effort to produce reproducible results